Loss of connexin45 causes a cushion defect in early cardiogenesis.

At around embryonic day 9, the primitive heart of a mouse embryo undergoes spectacular alterations within 24 hours. We created mice harboring an nls-lacZ gene in place of connexin45, which encodes the only known gap junction protein in the primitive heart before embryonic day 9, using the Cre-loxP system. Connexin45-deficient mice died of heart failure at around embryonic day 10. They initiated heart contractions, but conduction block appeared within 24 hours after the first contractions. Their cardiac walls displayed an endocardial cushion defect, while the cardiac jelly was present. These abnormalities were caused by impairment of the epithelial-mesenchymal transformation of the cardiac endothelium. Activation of the cardiac endothelium depended on the presence of the connexin45 gap junctions since signaling through Ca(2+)/calcineurin and NF-ATc1 (originally named NF-ATc) was disrupted in the mutant hearts. These results indicate a requirement for gap junction channels during early cardiogenesis and hence implicate connexin45 in congenital heart diseases. http://www. biologists.com/Development/movies/dev4369.html

Detection of allergen- and mitogen-induced human cytokine transcripts using a competitive polymerase chain reaction.

Human cytokines, IL-4, IL-5, and IFN-gamma play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-gamma) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-gamma transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS) cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector containing the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the 'competitive' PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-gamma transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finally, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-gamma, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.

Immunogenetic aspects of IgE-mediated responses.

The reported significant HLA association is consistent with the codominant expression of MHC-linked Ir genes. Similar studies are needed in the case of the other HLA-immune response associations. It seems unlikely that any unique HLA-D genetic sequence will be found only among subjects responding to a particular Ag. It is likely, however, that we will find that a particular sequence is a necessary, but not sufficient, requirement for responsiveness to a particular antigenic epitope. Previous family studies, in which failed to observed parent-to-child transmission of specific immune responsiveness to a particular allergen, suggest that further genetic and/or environmental factors are required for the expression of specific immune responsiveness. These factors include variations in the degree of antigenic exposure. Of particular importance is the need for Ag-specific TcR genes to be expressed in the mature T-cell repertoire. In study of the specific immune response, there have been several studies of the protein and DNA sequences of allergenic proteins; therefore, in regard to understanding the genetics of specific immune responsiveness to allergens and its relationship to atopic diseases, rapid advances can be anticipated over the next several years.

[Immunological study of the HLA class II antigen associated with birch pollen allergy].

Pollinosis is known as a hereditary disease. Recently, association of HLA antigens and pollinosis caused by several pollen allergens has been reported. Furthermore, HLA antigens are considered to have a very important role in the development of pollinosis. We performed an HLA population study and tested the lymphocyte proliferative response (LPR) to birch pollen allergens with birch pollen allergic patients and healthy control subjects. The results of the HLA population study indicated that HLA-DR9 and HLA-DQw3 correlated with the development of birth pollinosis (relative risk [R. R] = 6.37 for DR9 and R. R = 7.92 for DQw3). DNA typing revealed that serotype DQw3 in patients were DQw9 specific. Two sources of birch pollen allergens, betula pendula and betula platyla var. japonica, were used in LPR. Lymphocytes from the patients, but not from the healthy control subjects, proliferated in response to these two allergens. Anti-HLA-DR antibody, but not anti-HLA-DQ antibodies, inhibited the patients' LPR. These results suggest that the HLA-DR molecules are responsible to present the pollen specific antigen to T lymphocytes inducing LPR in the patients. Furthermore, in some of the healthy control subjects, anti HLA-DQ antibodies enhanced this LPR almost to the same level as that of the patients'. This result indicates that HLA-DQ molecules might be associated with the suppression of T cell proliferation.